The present invention relates to the field of biotechnology. More particularly, the invention pertains to the design of systems for cooling biological samples, and to the handling and positioning of sample holders to receive and store cold samples. The invention also pertains to the more general problem of accurately positioning objects that are immersed in cryogenic fluids.
Cryogenic Sample Cooling and Handling in X-Ray Crystallography:
X-ray crystallography is the most powerful and widely used tool for determining the molecular structures of proteins, viruses, nucleic acids, and biomolecular complexes. The determination of these molecular structures is critical to modern molecular biology, and to the development of pharmaceutical treatments for various diseases and human conditions.
X-ray data collection is typically performed on crystals cooled to T=100 K or below. At room temperature, crystals of proteins, viruses and other biomolecules are rapidly damaged by X-rays. In most cases, only a fraction of the data required to determine the molecular structure can be obtained from each crystal, especially when small (10-50 μm) crystals produced in early crystallization trials are used. Growing large crystals requires careful, expensive, and time-consuming optimization. At room temperature, crystals must be handled carefully: they can easily be damaged by mechanical contact; they can dry out, causing loss of diffraction; and significant temperature changes can cause changes in protein structure. Thus, more than 98% of X-ray data is collected from crystals cooled to T˜100 K. At cryogenic (e.g., T<120 K) temperatures, crystals can typically withstand X-ray doses 30-1000 times larger than at 300 K, sometimes allowing structure determination using a single crystal. Crystals are cooled by plunging them from air into liquid nitrogen or liquid propane. Cryo-cooled crystals are as hard as ice and insensitive to large temperature changes as long as the temperature remains below ˜150 K, so that they can be easily stored, shipped, and manipulated at the synchrotron. Consequently, the introduction of cryocrystallographic methods for biomolecular crystallography in the 1990s was transformative.
In typical cryocrystallographic practice, a crystal is either grown in or soaked in a drop containing cryoprotectants (glycerol, ethylene glycol, 2-methyl-2,4-pentanediol (MPD), polyethylene glycol (PEG)), and harvested from the drop using a nylon or microfabricated loop attached to a metal goniometer base. It is then directly plunged by hand into an open-mouth Chamber or an open foam box filled with liquid nitrogen (in the process bringing typically ungloved fingers within centimeters of the liquid cryogen). To facilitate organization, storage, shipping, and sample management at the X-ray source, cryo-cooled samples are usually inserted into sample “carousel”, “puck”, or “cassette” that accepts many samples. This multiple sample holder is typically immersed in the same liquid nitrogen as is used to cool the samples, so that samples are transferred to the holder without risk of warming.
Once loaded in cryovials or multiple sample pucks, the samples are stored in cryogenic chambers or dry shippers. These are transported to an X-ray source, which may be a commercial X-ray system in the home laboratory, or a synchrotron X-ray source at a national facility. At the X-ray source, samples in pucks are loaded into a liquid-cryogen-filled container of an automated sample changer, and the automated changer then selects, pulls out of the puck, and positions each sample for X-ray examination. When X-ray measurements on a sample are complete, the automated changer returns the sample to the puck, and selects another sample for measurement. In these sample changer devices, the pucks may be stationery, and samples selected by a moving arm, or the sample holding arm may be in a fixed position, and the desired sample is translated or rotated beneath the arm.
More recently, an automated system for plunge cooling of protein crystals has been developed. In such systems, automated loading of cryogenically cooled samples into cryogenically cooled sample holding pucks/cassettes is highly desirable.
Cryogenic Sample Cooling and Handling in Cryoelectron Microscopy and in Cryopreservation of Biomolecules, Cells, and Tissues:
Aside from crystallography, many other applications involve the cryogenic cooling, storage, and subsequent retrieval of biological samples, including samples for cryoelectron microscopy, for cold storage of proteins and biologic drugs, and for cryopreservation of cells (e.g., gametes, stem cells) and tissues. In all of these applications, samples may be cooled by plunging in a liquid cryogen (liquid propane or ethane in cryoelectron microscopy, liquid nitrogen in other applications), and then transferred to a multiple sample holder that is immersed in liquid nitrogen. Multiple sample holders can then be transferred to a cryogenic temperature dry storage container. Sample cooling and transfer to the sample holder is typically done by hand, but there is increasing interest in automated systems that can improve reproducibility and reliability.
Methods for Transferring and Storing Samples Immersed in Liquid Cryogens:
Several methods are used for automated transfer of samples to, and from multiple sample holders/cassettes immersed in liquid cryogens. In one method, the multiple sample holder is held in fixed position within a liquid nitrogen containing chamber, and individual samples loaded and retrieved using a robotic arm with full x-y-z motion that accesses the samples from above the chamber. In a second method, the robotic arm is replaced by a simple vertical translation (z) arm mounted on an x-y stage, again placed above the liquid nitrogen containing chamber.
A major limitation of these approaches is that a large area of liquid nitrogen surface—at least the area of the multiple sample holder—may be exposed to ambient air, causing excessive boil-off of liquid nitrogen and accumulation of frost on cold surfaces and in the liquid nitrogen, contaminating the liquid nitrogen, and possibly also the sample. This can be addressed by using relatively complicated mechanisms such as x-y sliding chamber covers with small openings that can be translated laterally to expose each sample position, but these covers are large and bulky.
Achieving the positional accuracy required to place large numbers of samples into holders and to retrieve them is not trivial, especially if the chamber undergoes thermal cycling and/or has not reached steady state dimensions.
A potentially simpler approach that can minimize exposure of the liquid nitrogen to ambient air, minimize boil-off, and minimize accumulation of ice is to translate samples only along a single axis (rather than in x, y and z axes), that passes through a small fixed opening in a lid that covers and insulates the liquid nitrogen chamber. Samples can be translated along this axis into the liquid nitrogen for cooling, and then translated along this axis within the liquid nitrogen and inserted into a sample holder.
This approach requires a mechanism that operates in liquid nitrogen for holding samples in one or more cassettes, each with multiple sample positions, and for positioning one or multiple cassettes each with multiple sample positions so as to place a particular sample position in a particular sample cassette directly along the sample translation axis, so that samples can be loaded and retrieved with a simple single axis linear motion. Sample holder cassettes could then be removed, cryogenically stored, shipped to another location, and transferred into a liquid nitrogen chamber with a similar sample holder cassette positioner, vertical (or tilted) sample translation axis, and a small access hole in a chamber cover, from which samples could be retrieved.